Apicomplexan parasites are obligate intracellular protozoa that cause severe human diseases including malaria, cryptosporidiosis, and toxoplasmosis. These parasites must invade host cells to survive, proliferate, and cause disease. There is mounting evidence that parasite secretory proteins play key roles in invasion. The long-term goal of the parent R01 proposal is to elucidate the function of parasite secretory organelles called micronemes, using Toxoplasma gondii as a model parasite for other Apicomplexa. These studies will be conducted in an effort to identify novel targets for anti-parasitic treatments. While the parent proposal focused exclusively on one protein, MIC2, in this R21 we plan to expand the scope of our analysis to include two additional microneme proteins, MIC5 and MIC2-associated protein (M2AP), which forms a heterodimer with MIC2. This expansion will be accomplished using several powerful, cutting-edge technologies including gene knockouts, high-resolution separation and mass spectrometry identification of proteins, and affinity purification of protein complexes. Specific Aim 1 of this R21 proposal is to define the impact of MIC5, M2AP, or MIC2 gone deletions on the expression, secretion, post-translational modifications, and targeting of all other proteins secreted by T. gondii. Specific Aim 2 is to identify the protein partner(s) for MIC5 that are responsible for targeting MIC5 to the micronemes and then to the parasite surface during invasion. We anticipate that this "functional proteomics" project will reveal previously unknown protein interactions that will be pivotal in defining the functions of secretory proteins in parasite invasion.